Stabilized albumin preparations

ABSTRACT

This invention provides albumin preparations with safety and without any risk of side effects, which are free from viruses or contaminating proteins and can be stably stored over a long time while showing neither changes in appearance nor decrease in content. There are provided a stabilized albumin preparation produced by uniformly mixing a medium-chain fatty acid or a salt thereof and a sulfur-containing amino acid or a derivative thereof with an aqueous albumin solution (e.g., a buffer such as phosphate buffer which can be administered as pharmaceutical preparations, injection water, or a physiological saline) and dissolving them therein, and then processing the mixture solution into a formulation suitable for parenteral administration such as an intravenous fluid preparation or an injectable solution, and a stabilization method for an albumin preparation.

TECHNICAL FIELD OF THE INVENTIONI

present invention relates to albumin preparations having excellentstability, and more particularly to albumin preparations such as thoseof a fractionated human serum albumin and a genetically engineered humanserum albumin, which have excellent stability as well as safety.

BACKGROUND ART

For diseases based on acute hypoalbuminemia and chronic hypoalbuminemiawhich are hard to control, albumin preparations are used for improvingclinical conditions by the supplementation thereof. Specifically, theyare medical supplies indispensable for modern medical treatment becausethey are generally Used for correction of a circulating plasma volume inhemorrhagic and traumatic shock, improvement of edema, and various kindsof diseases such as liver cirrhosis and nephrotic syndrome (Peter T.Jr., The Plasma Proteins. Academic Press. New York. 133-81 (1975),Rosenorer V M., Rothschild M A., Albumin Structure, Function and Uses(1977)).

Conventionally, the production of human serum albumin preparations hasbeen performed by fractionating blood collected from a human being andpurifying the obtained albumin-containing aqueous solution according tovarious kinds of purification methods. The purification methods includean ethanol fractionation method, a PEG fractionation method, an ammoniumsulfate fractionation method, a method in which the use of an anionexchanger is combined with heat treatment at 69° C. for 10 hours (JP2-191226A), and a method in which treatment with an anion exchanger,treatment with a cation exchanger, and heat treatment at 60° C. for 10hours are combined together (JP 3-17123A and JP 7-330626A). On the otherhand, in recent years, the technology for mass production of albuminwith a recombinant (gene recombination) has been established, and thusit has become possible to produce human albumin in large quantity byfactory production without depending on blood donation (ClinicalMolecular Medicine, 1, 939 (1993)).

In the production of albumin preparations (particularly, the productionof fractionated human serum albumin), for removing a harmful virusunstable to heat and preventing contamination with a protein or thelike, for example, a sterilization method such as low-temperaturesterilization (60° C. for 10 hours) is used. In the low-temperaturesterilization, N-acetyl tryptophan and sodium caprylate are added tohuman serum albumin (Ballou G A., Boyer P D., Luck J M., Lum F G., J.Biol. Chem., 153, 589-605 (1944), Scatchard G, Strong L E., Hughes W L.Jr., Ash Worth J N., Sparrow A H., J Clin. Invest., 24, 571-679 (1945),Boyer P D., Lum F G., Ballou G A., Luch J M., Rice R G., J.Biol. Chem.,162, 181-198 (1946)). However, the N-acetyl tryptophan has a side-effectproblem such as intracerebral disease.

It is intended to provide albumin preparations with safety and withoutany risk of side effects, which are free from viruses or contaminatingproteins and can be stably stored over a long time while showing neitherchanges in their appearance nor decrease in their content.

DISCLOSURE OF THE INVENTION

Under such circumstances, the inventors of the present invention havefound that N-acetyl methionine exerts an excellent effect of stabilizingalbumin and discovered that the N-acetyl methionine has an excellenteffect of depressing agglomeration by coexisting with a medium-chainfatty acid. As a result of various studies on the basis of thosefindings, the present invention has been completed.

More specifically, the present invention relates to the followingitems 1) to 8):

-   1) A stabilized albumin preparation, characterized by comprising a    medium-chain fatty acid or a salt thereof and a sulfur-containing    amino acid or a derivative thereof;-   2) The albumin preparation as claimed in item 1, wherein the    medium-chain fatty acid is a straight-chain saturated fatty acid    having 6 to 12 carbon atoms;-   3) The albumin preparation as claimed in item 1, wherein the    sulfur-containing amino acid is an amino acid having a mercapto    group which may optionally be alkylated or may optionally be    dimerized to a disulfide bond;-   4) The albumin preparation as claimed in item 1, wherein the    sulfur-containing amino acid derivative is an N-acylated    sulfur-containing amino acid derivative;-   5) The albumin preparation as claimed in item 1, wherein the total    addition amount of the medium-chain fatty acid or a salt thereof and    the sulfur-containing amino acid or the derivative is an    approximately 1- to 20-fold molar quantity of albumin;-   6) The albumin preparation as claimed in Claim 1, wherein the    albumin is a genetically engineered human serum albumin;-   7) A stabilizer for an albumin preparation, which comprises a    medium-chain fatty acid or a salt thereof and a sulfur-containing    amino acid or a derivative thereof; and-   8) A stabilization method for an albumin preparation, characterized    by comprising blending a medium-chain fatty acid or a salt thereof    and a sulfur-containing amino acid or a derivative thereof.

In preparations according to the present invention, fractionated humanserum albumin, genetically engineered human serum albumin, or the likeis used as the effective ingredient albumin. The albumin preparation isused in the form of a solution generally having an albumin content ofapproximately 5 w/v % to approximately 25 w/v %. Solvents for dissolvingalbumin include water (injection water), a physiological saline, andbuffers such as phosphate buffer which can be administered aspharmaceutical preparations.

Examples of medium-chain fatty acids to be added to the preparations ofthe present invention include fatty acids having 6 to 12 carbon atoms,preferably straight-chain saturated fatty acids having 6 to 12 carbonatoms. Preferable specific examples include caprylic acid, pelargonicacid, capric acid, undecanoic acid, and lauric acid. The salts ofmedium-chain fatty acids include basic salts such as alkali metal salts,for example, sodium salt and potassium salt. Among them, sodiumcaprylate is most preferable.

The sulfur-containing amino acids or derivatives thereof include aminoacids having mercapto groups (SH groups) which may optionally bealkylated or may optionally be dimerized (a disulfide bond). Thesulfur-containing aminoacids include amino acids having 1 to 3 sulfuratoms in a molecule. Preferable specific examples thereof includecysteine, cystine, and methionine. The derivatives of sulfur-containingamino acids include N-acyl derivatives, preferably, for example,N-(alkanoyl having 1 to 6 carbon atoms) derivatives such as N-formyl,N-acetyl, N-propionyl, and N-butyryl, and, more preferably, N-acetylderivatives. Among them, N-acetyl methionine is most preferable.

In the preparations of the present invention, the total addition amountsof the medium-chain fatty acid or the salt thereof and thesulfur-containing amino acid or the derivative thereof is preferablyabout an equimolar quantity to 20-fold molar quantity of albumin.Addition molar ratio of the added sulfur-containing amino acid or thederivative thereof: the added medium-chain fatty acid or the saltthereof is 0.1 to 10:1, preferably 0.5 to 2:1. One kind or two or morekinds of each of the medium-chain fatty acids or the salts thereof andthe sulfur-containing amino acids or the derivatives thereof may besuitably mixed together in use.

In the preparations of the present inventions, if needed, a suitableamount of a pharmaceutically acceptable additive such as a colorant, astabilizer, an antiseptic, a diluent, a pH regulator (e.g., basic aminoacid, acidic amino acid, hydrochloric acid, acetic acid, malic acid, orsodium hydroxide), an osmolality regulator (e.g., an electrolyte such assodium chloride, potassium chloride, potassium gluconate, magnesiumsulfate, sodium bicarbonate, calcium chloride, calcium gluconate, orcitric acid), or a surfactant (e.g., nonionic surfactant) may be added.

The pH value of a solution of the preparations of the present inventionis adjusted to about pH 5 to 7.5, preferably about pH 6.5 to 7.4 by theaddition of a pH regulator if required. The preparations of the presentinvention may further contain a physiologically active substance otherthan albumin when needed. For instance, saccharides (e.g.,monosaccharides such as glucose and fructose, disaccharides such asmaltose, and sugar alcohols such as sorbitol and xylitol) may be added.

The preparations of the present invention are used for therapeuticpurposes, such as correction of a circulating plasma volume inhemorrhagic and traumatic shock, improvement of edema, and various kindsof diseases such as liver cirrhosis and nephrotic syndrome. They aregenerally administered to an adult in a dose of about 5 to 12.5 g at atime. The preparations of the present invention may be administered onceor in about two to four portions a day depending on the diseasecondition.

The preparations of the present invention have almost no side effectswith lower toxicity and safety, and they can be subcutaneously orintravenously administered to human beings and mammals (e.g., sheep,horses, and cows) according to methods known per se.

The preparations of the present invention can be prepared by uniformlymixing the medium-chain fatty acid or the salt thereof and thesulfur-containing amino acid or the derivative thereof with an aqueousalbumin solution (e.g., a buffer such as phosphate buffer which can beadministered as pharmaceutical preparations, injection water, or aphysiological saline) and dissolving them therein, and thenpharmaceutically processing the mixture solution into a formulationsuitable for parenteral administration, such as intravenous fluidpreparation or an injectable solution.

The medium-chain fatty acid or the salt thereof, the sulfur-containingamino acid or the derivative thereof, and albumin, which are used as rawmaterials, can be prepared by conventional methods or methods known perse.

Vessels for storing the resulting preparations include a glass vial anda plastic vessel made of polypropylene, polyethylene, or the like. Thealbumin preparations of the present invention can be filled in the abovevessels or the like and hermetically sealed, and then subjected tosterilization (60° C./10 hours).

The preparations of the present invention show excellent stabilitybecause of little change in appearance and substantially no change incontents thereof even after sterilization under heat or long storage.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 An alternative photograph in place of a drawing for showingundenatured polyacrylamide gel electrophoresis of Experimental Example2.

FIG. 2 An alternative photograph in place of a drawing for showingundenatured polyacrylamide gel electrophoresis of Experimental Example3.

FIG. 3 A diagram showing an HPLC chromatogram of Experimental Example 4.

FIG. 4 A diagram showing carbonyl content in HSA with oxidation ofExperimental Example 5.

THE BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be explained concretely withreference to examples and experimental examples, but not limited tothose examples.

EXAMPLE 1

Genetically engineered human serum albumin (hereinafter, human serumalbumin will be abbreviated as HSA or albumin) was dissolved in aphysiological saline to prepare 500 mL of an aqueous 25-w/v % HSAphysiological saline solution. Then, 1662 mg of sodium caprylate and1912.5 mg of N-acetyl methionine were added as stabilizers and dissolvedtherein, followed by hermetically sealing after dispensing the aqueousHSA physiological saline solution in an amount of 50 mL each into 50-mLvials.

EXAMPLE 2

HSA fractionated from blood was dissolved in a physiological saline toprepare 500 mL of an aqueous 25-w/v % HSA physiological saline solution.Then, 1662 mg of sodium caprylate and 1912.5 mg of N-acetyl methioninewere added as stabilizers and dissolved therein, followed byhermetically sealing after dispensing the aqueous HSA physiologicalsaline solution in an amount of 50 mL each into 50-mL vials.

EXPERIMENTAL EXAMPLE 1

1) Preparation of Medical Agent Under Test

Degreasing is performed on genetically engineered HSA by the method ofChen (Chen R F, J. Biol. Chem., 242, 173-181 (1967)). Furthermore, itwas freeze-dried after dialysis and then used in all of the experimentsdescribed below.

Each of various medical agents was added to 100-μM HSA ( 1/15 mMphosphate buffer, pH 7.4) and then the following medical agents undertest were prepared:

(1) a 100-μM HSA solution;

(2) a solution of 100-μM HSA+500-μM sodium caprylate;

(3) a solution of 100-μM HSA+500-μM N-acetyl methionine; and

(4) a solution of 100-μM HSA+500-μM N-acetyl methionine+500-μM sodiumcaprylate.

2) Measuring Method

The stabilization effect was evaluated in terms of a thermodynamicviewpoint by calculating a thermal transition temperature (Tm) and achange ΔHcal in enthalpy of transition from a differential scanningcalorimetry (DSC) measurement on those medical agents under test (1) to(4). If the thermal transition temperature (Tm) increases, from thethermodynamic viewpoint, it means an increase in stability of themedical agents under test. In addition, if the change ΔHcal in enthalpyof transition increases, it means an increase in stability of themedical agents transition under test.

In Table 1, values of the thermal transition temperatures Tm, and valuesof the changes ΔHcal in enthalpy of transition of the respective medicalagents under test (1) to (4) are shown. Those values were analyzed by aDSC thermogram of the HSA solution. Differential scanning calorimetry(DSC method)

For the DSC, measurement was made using the MC-2 differential scanningcalorimeter manufactured by MicroCal Co., Ltd. The measuring conditionsare shown below.

Scanning rate: 1 K/min.

Albumin content: 100 μM Solvent: 1/15 M phosphate buffer (pH 7.4)

Reversibility of thermal denaturation was confirmed by cooling thealbumin solution after first measurement and then heating the albuminsolution again. As a result, it was found that the albumin causedreversible thermal transition without bringing about irreversibledenaturation as far as it was heated up to 85° C. or less. The resultingthermogram was subjected to fitting using a nonlinear fitting algorithm(Using Origin TM scientific plotting software), followed by analysis asdescribed below by the use of a lower area of the resulting excessthermal capacity curve.Change in enthalpy of transition ΔHcal=∫Cex dTwherein Cex represents an excess thermal capacity.3) Results

As is evident from Table 1, an increase in thermal transitiontemperature (Tm) was confirmed in each of the solutions with theadditions of various medical agents, compared with an HSA solutionwithout the addition of any medical agent. The addition of sodiumcaprylate leads to about a 7° C. increase in Tm of albumin, so that anincrease in stability thereof to heat was shown.

In addition, the addition of N-acetyl methionine alone allowed a slightincrease in Tm and an increase in change ΔHcal in enthalpy transition.

The solution added with sodium caprylate and N-acetyl methionine was ina conformational state in which albumin was placed in a more methodicalfashion, and thus it was found that the state is more stable than thenative state. TABLE 1 Medical Agent under test Tm (° C.) Δhcal(10⁵kcal/mol) (1) HSA 59.50 1.64 (2) HSA + sodium caprylate 66.83 2.1(3) HSA + N-acetyl methionine 60.60 1.81 (4) HSA + sodium caprylate +66.51 2.03 N-acetyl methionine

EXPERIMENTAL EXAMPLE 2

1) Preparation of Medical Agents Under Test

A 24-μM HSA phosphate buffer (67 mM, pH 7.4) was prepared and variousmedical agents were added thereto so as to have concentrations of 24,72, or 120 μM to make medical agents under test (1) to (4) for examiningthe influence on the concentration of the medical agent added on thestabilization effect:

(1) 24-μM HSA;

(2) 24-μM HSA+sodium caprylate;

(3) 24-μM HSA+N-acetyl methionine; and

(4) 24-μM HSA+N-acetyl methionine+sodium caprylate.

2) Measuring Method

The thermal stability with sterilization under heat at 60° C. for 30minutes was evaluated by means of NATIVE-PAGE. NATIVE-PAGE

NATIVE-PAGE was conducted using a 7.5% (w/v) polyacrylamide gel inaccordance with the Davis method. Proteins were stained with Coomassiebrilliant blue R-250 and the following proteins were used as molecularweight markers:

α-thyroglobulin (M. W. 669000), ferritin (M. W. 443000), lactatedehydrogenase (M. W. 139850), bovine serum albumin (M. W. 66267), andtrypsin inhibitor (M. W. 20110) were used.

3) Results

The results of NATIVE-PAGE are shown in FIG. 1. After the heat treatmentat 60° C. for 30 minutes, a large quantity of aggregates was confirmedin the medical agent under test (1) (no medical agent added). On theother hand, the aggregate-inhibition effects were confirmed in themedical agent under test (2) (containing sodium caprylate), the medicalagent under test (3) (containing N-acetyl methionine), or the medicalagent under test (4) (containing sodium caprylate and N-acetylmethionine), respectively. With respect to their concentrationdependence, as was evident from migration patterns, almost noaggregate-inhibition effect was confirmed when the medical agent wasadded at an equimolar concentration to albumin. However, a distinctaggregate-inhibition effect was observed by the addition in 3-foldconcentration (72 μM) and a remarkable aggregate-inhibition effect wasobserved by the addition in 5-fold concentration (120 μM).

EXPERIMENTAL EXAMPLE 3

The aggregate-inhibition effect under heat was examined usingmedium-chain fatty acids except sodium caprylate (electrophoresis inFIG. 2). The measurement samples used were such that 5-foldconcentrations (120 μM) of the respective fatty acids were each added toa 24-μM HSA phosphate buffer (67 mM, pH 7.4).

As a result, the aggregate-inhibition effect was slightly observed incaproic acid (C6) and enanthic acid (c7) having a carbon chain shorterthan that of sodium caprylate and remarkable inhibition effects wereobserved in fatty acids having carbon chains not shorter than that ofsodium caprylate, respectively.

EXPERIMENTAL EXAMPLE 4

1) Preparation of Medical Agents Under Test A 24-μM HSA phosphate buffer(64 mM, pH 7.4) was prepared and then medical agents under test wereprepared as described below by the addition of various medical agents ata concentration of 120 μM, followed by adding2,2′-azobis-(2-amidino-propane) dihydrochloride (hereinafter referred toas AAPH) (first grade, manufactured by Wako Pure Chemical Industries,Ltd.) to 5 mL of the medical agent under test so as to have a finalconcentration of 10 μM:

(1) HSA;

(2) HSA+sodium caprylate; and

(3) HSA+sodium caprylate+N-acetyl methionine

2) Measurement of Abundance Ratio Between Mercapto Type and Non-MercaptoType of HSA

The abundance ratio (mercapto fraction) between the mercapto type andnon-mercapto type of HSA was determined using high performance liquidchromatography (hereinafter, referred to as HPLC).

The device for HPLC used for the measurement of mercapto fraction wasthe Shimadzu LC-4A equipped with a gradient device and connected to theShimadzu SPD-24SUV detector and the Shimadzu C-R2AX data-processingdevice. Elution of the sample was performed using a method of a lineargradient from (A) a 0.05-M tris-acetate buffer (pH 7.0) to (B) a 0.05-Mtris-acetate buffer (pH7.0) containing 0.5-M sodium acetate for 30minutes at a flow rate of 0.5 mL/min. The sample was detected using280-nm UV at room temperature.

3) Results

A HPLC chromatogram of HSA in the medical agent under test (1) is shownin FIG. 3. In the medical agent under test (1), a decrease in mercaptotype and an increase in non-mercapto type were observed.

A change in abundance ratio between the mercapto type and non-mercaptotype of HSA could not be confirmed even after the addition of sodiumcaprylate. However, the medical agent under test (3) (concomitant use ofsodium caprylate and N-acetyl methionine) inhibits a change in abundanceratio therebetween significantly and an antioxidative effect wasconfirmed. Therefore, it is concluded that N-acetyl methionine withoutside effects is suitable as a stabilizer.

EXPERIMENTAL EXAMPLE 5

An excess amount of an oxidizing agent, 2,2′-azobis-(2-amidinopropane)dihydrochloride (AAPH) was added to HSA to oxidize the HSA. After theoxidation for a predetermined time, the carbonyl content in HSA wasdetermined. From the relationship between the oxidizing time and thetype of a stabilizer, the anti-oxidation actions of various stabilizerswere evaluated (FIG. 4). The carbonyl content, which indicates thedegree of oxidation, was determined in accordance with the method ofCliment et al. (described in Climent I., Thai L., and Levine R L., AnalBiochem. 182, 226 (1989)) and represented as a modification amount perprotein of a carbonyl-coloring reagent, Fluorsceinamine.

In the case of N-acetyl methionine, a significant decrease in carbonylcontent was observed in comparison with the medical agent under testwithout addition. Thus, it is clear that the anti-oxidation action canbe obtained.

EXPERIMENTAL EXAMPLE 6

Using a physiological saline and genetically engineered HSA, 500 mL of a25-w/v % solution was prepared. Then, 1662 mg of sodium caprylate and1912.5 mg of N-acetyl methionine were added as stabilizers thereto toprepare the medical agents under test, followed by hermetically sealing50 mL of the agent in a vial. In addition, the genetically engineeredHSA without a stabilizer was prepared with a physiological saline in aconcentration of 25 w/v %, followed by hermetically sealing 50 mL of theresultant in a vial.

Those samples were subjected to heat treatment under sterilizationconditions of 60° C. for 30 minutes, and then the generation ofcontaminants was visually observed. As a result, in the sample withoutthe addition of the additive, the generation of albumin aggregates byheat was distinctly observed. On the other hand, no contaminant wasobserved in the sample with the addition of the stabilizer.

INDUSTRIAL APPLICABILITY

The stabilized albumin preparations of the present invention can besafely stored for a long term in a stable manner without risk of sideeffects. In the case of the addition of medium-chain fatty acid or thesalt thereof alone, a thermal stabilization effect can be observed butthe anti-oxidization action of albumin is small. In the case of theaddition of sulfur-containing amino acid or the derivative thereofalone,no thermal stabilization effect on albumin is observed. Therefore,the addition of both the medium-chain fatty acid or the salt thereof andthe sulfur-containing amino acid or the derivative thereofsynergistically provides an excellent albumin stabilization effect.

1. A stabilized albumin preparation, characterized by comprisingalbumin, a medium-chain fatty acid or a salt thereof and asulfur-containing amino acid or a derivative thereof.
 2. The albuminpreparation as claimed in claim 1, wherein the medium-chain fatty acidis a straight-chain saturated fatty acid having 6 to 12 carbon atoms. 3.The albumin preparation as claimed in claim 1, wherein thesulfur-containing amino acid is an amino acid having a mercapto groupwhich may optionally be alkylated or may optionally be dimerized to adisulfide bond.
 4. The albumin preparation as claimed in claim 1,wherein the sulfur-containing amino acid derivative is an N-acylatedsulfur-containing amino acid derivative.
 5. The albumin preparation asclaimed in claim 1, wherein the total addition amount of themedium-chain fatty acid or a salt thereof and the sulfur-containingamino acid or the derivative thereof is an approximately 1- to 20-foldmolar quantity of albumin.
 6. The albumin preparation as claimed inclaim 1, wherein the albumin is a genetically engineered human serumalbumin.
 7. A stabilizer for an albumin preparation, which comprises amedium-chain fatty acid or a salt thereof and a sulfur-containing aminoacid or a derivative thereof.
 8. A stabilization method for an albuminpreparation, characterized by comprising blending a medium-chain fattyacid or a salt thereof and a sulfur-containing amino acid or aderivative thereof with albumin.